This invention describes carrier-drug conjugates prepared from disulfide analogs of the calicheamicin family of potent antitumor antibiotics and their derivatives, as well as similar analogs from related antitumor antibiotics such as the esperamicins. The carrier can be an antibody, growth factor, or...http://www.google.com/patents/US5877296?utm_source=gb-gplus-sharePatent US5877296 - Process for preparing conjugates of methyltrithio antitumor agents

Process for preparing conjugates of methyltrithio antitumor agentsUS 5877296 A

Abstract

This invention describes carrier-drug conjugates prepared from disulfide analogs of the calicheamicin family of potent antitumor antibiotics and their derivatives, as well as similar analogs from related antitumor antibiotics such as the esperamicins. The carrier can be an antibody, growth factor, or steroid which targets an undesired population of cells, such as those of a tumor. Whole protein carriers as well as their antigen-recognizing fragments and their chemically or genetically manipulated counterparts are useful for the targeting portion of the conjugates. This invention includes compounds required for the synthesis of these conjugates, appropriate pharmaceutical compositions of the carrier-drug conjugates, and their method of use.

Images(29)

Claims(15)

We claim:

1. A process for preparing the targeted derivatives of formula

Z3 (CO-Alk1 -Sp1 -Ar-Sp2 -Alk2 -C(Z1)=Z2)m

wherein

Z3 is a protein selected from mono- and polyclonal antibodies, their antigen-recognizing fragments, and their chemically or genetically manipulated counterparts;

Alk1 and Alk2 are independently a bond or branched or unbranched (C1 -C10) alkylene chain;

Sp1 is a bond, --S--, --O--, --CONH--, --NHCO--, --NR'--, --N(CH2 CH2)2 N--, or -X-Ar'-Y-(CH2)n -Z wherein X, Y, and Z are independently a bond, --NR'--, --S--, or --O--, with the proviso that when n=0, then at least one of Y and Z must be a bond and Ar' is 1,2-, 1,3-, or 1,4-phenylene optionally substituted with one, two, or three groups of (C1 -C5) alkyl, (C1 -C4) alkoxy, (C1 -C4) thioalkoxy, halogen, nitro, COOR', CONHR', O(CH2)n COOR', S(CH2)n COOR', O(CH2)n CONHR', or S(CH2)n CONHR', with the proviso that when Alk1 ' is a bond, Sp1 ' is a bond;

n is an integer from 0 to 5;

R' is a branched or unbranched (C1 -C5) chain optionally substituted by one or two groups of --OH, (C1 -C4) alkoxy, (C1 -C4) thioalkoxy, halogen, nitro, (C1 -C3) dialkylamino, or (C1 -C3) trialkylammonium-A- where A- is a pharmaceutically acceptable anion completing a salt;

Ar is 1,2-, 1,3-, or 1,4-phenylene optionally substituted with one, two, or three groups of (C1 -C6) alkyl, (C1 -C5) alkoxy, (C1 -C4) thioalkoxy, halogen, nitro, or COOR', CONHR', O(CH2)n COOR', S(CH2)n COOR', O(CH2)n CONHR', or S(CH2)n CONHR' wherein n and R' are as defined above or a 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-, 1,8-, 2,3-, 2,6-, or 2,7-naphthylidene or ##STR16## each naphthylidene or phenothiazine optionally substituted with one, two, three, or four groups of (C1 -C6) alkyl, (C1 -C5) alkoxy, (C1 -C4) thioalkoxy, halogen, nitro, COOR', CONHR', O(CH2)n COOR', S(CH2)n COOR', O(CH2)n CONHR', or S(CH2)n CONHR' wherein n and R' are as defined above, with the proviso that when Ar is naphthylidene, Z1 is not hydrogen and with the proviso that when Ar is phenothiazine, Sp1 is a bond only connected to nitrogen;

Sp2 is a bond, --S--, or --O--, with the proviso that when Alk2 is a bond, Sp2 is a bond;

in an alcoholic solvent with a boiling point of less than about 100° C. in the presence of about 5% acetic acid or a carboxylic acid catalyst at about 20° to 70° C. for about 1 to 24 hours, wherein Alk1 and Alk2, Sp1, n, R', Sp2, Z1, and Ar are as defined above, to produce an intermediate of formula

HOCO-Alk1 -Sp1 -Ar-Sp2 -Alk2 -C(Z1)═Z2,

wherein Alk1, Sp1, Ar, Sp2, Alk2, Z1, and Z2 are as defined above;

(b) isolating the intermediate of step (a);

(C) reacting the isolated intermediate of step (b) with N-hydroxysuccinimide, 2, 3, 5, 6-tetrafluorophenol, pentafluorophenol, 4-nitrophenol, 2,4-dinitrophenol, or N-hydroxysulfosuccinimide in the presence of DCC, EDCI, or other carbodiimide in an inert organic solvent such as acetonitrile or acetonitrile containing 5-50% DMF to generate the compound

Z3' CO-Alk1 -Sp1 -Ar-Sp2 -Alk2 -C(Z1)═Z2,

wherein Alk1, Sp1, Ar, Sp2, Alk2, Z1, and Z2 are as defined above; and Z3 is ##STR18## and (d) reacting the compound generated in step (C) of formula

Z3 CO-Alk1 -Sp1 -Ar-Sp2 -Alk2 -C(Z1)═Z2

with a carrier Z3, wherein Z3 is a protein selected from mono- and polyclonal antibodies, their antigen-recognizing fragments, and their chemically or genetically manipulated counterparts, in an aqueous, buffered solution at a pH of between 6.5 and 9.0 and a temperature of 4° to 40° C. for 1 to 48 hours to generate the targeted derivatives of formula

Z3 (CO-Alk1 -Sp1 -Ar-Sp2 -Alk2 -C(Z1)═Z2)m

defined above.

2. The process of claim 1, wherein Alk2 and Sp2 are together a bond and Z1 is H or (C1 -C5) alkyl.

3. The process of claim 2, wherein Sp1 is a bond, --S--, --O--, --CONH--, --NHCO--, or --NR', with the proviso that when Sp1 is a bond, Alk1 is a bond.

5. The process of claim 4, wherein a covalent bond to the Z3 protein is an amide formed from a reaction with the lysine side chains of the Z3 protein.

6. The process of claim 5, wherein Z2 is Q-Sp-S-S-W and W is ##STR19## R5, X, R5 ', R, and Sp are as defined in claim 1, and Q is ═NHNCO--.

7. The process of claim 6, wherein the alcoholic solvent of step

(a) is methanol; the carboxylic acid catalyst of step (a) is 5% acetic acid; the isolated intermediate of step (b) is reacted in step (C) with N-hydroxysuccinimide in the presence of EDCI in acetonitrile; and the aqueous buffered solution of step (d) is phosphate buffer having a pH of 7.4 to 8.0.

15. The process of claim 4, wherein Sp1 is --O--or a bond; Alk1 is a bond or branched or unbranched (C1 -C10) alkylene chain, with the proviso that when Alk1 is a bond, Sp1 is a bond; Z1 is (C1 -C5) alkyl; and Z2 is calicheamicin N-acetyl gamma dimethyl hydrazide.

Description

This is a divisional of application Ser. No. 08/253,877 filed on Jun. 3, 1994 now U.S. Pat No. 5,773,001.

SUMMARY OF THE INVENTION

This invention describes carrier-drug conjugates prepared from disulfide analogs of the calicheamicin family of potent antitumor antibiotics and their derivatives, as well as similar analogs from related antitumor antibiotics such as the esperamicins. The carrier can be an antibody, growth factor, or steroid which targets an undesired population of cells, such as those of a tumor. Whole protein carriers as well as their antigen-recognizing fragments and their chemically or genetically manipulated counterparts are useful for the targeting portion of the conjugates. This invention includes compounds required for the synthesis of these conjugates, appropriate pharmaceutical compositions of the carrier-drug conjugates, and their method of use.

More specifically, one aspect of the invention includes a cytotoxic drug conjugate of the formula:

Z3 CO-Alk1 -Sp1 -Ar-Sp2 -Alk2 -C(Z1)=Z2 !m

wherein

Z3 is a protein selected from mono- and polyclonal antibodies, their antigen-recognizing fragments, and their chemically or genetically manipulated counterparts and growth factors and their chemically or genetically manipulated counterparts, wherein a covalent bond to the protein is an amide formed from reaction with lysine side chains, or a steroid, wherein the covalent bond to the steroid is an amide or an ester;

m is from about 0.1 to 15;

Alk1 and Alk2 are independently a bond or branched or unbranched (C1 -C10) alkylene chain;

Sp1 is a bond, --S--, --O--, --CONH--, --NHCO--, --NR'--, --N(CH2 CH2)2 N--, or --X--AR'--Y--(CH2)n --Z wherein X, Y, and Z are independently a bond, --NR'--, --S--, or --O--, with the proviso that when n=0, then at least one of Y and Z must be a bond and AR' is 1,2-, 1,3-, or 1,4-phenylene optionally substituted with one, two, or three groups of (C1 -C5) alkyl, (C1 -C4) alkoxy, (C1 -C4) thioalkoxy, halogen, nitro, COOR', CONHR', O(CH2)n COOR', S(CH2)n COOR', O(CH2)n CONHR', or S(CH2)n CONHR', with the proviso that when Alk1 is a bond, Sp1 is also a bond;

n is an integer from 0 to 5;

R' is a branched or unbranched (C1 -C5) chain optionally substituted by one or two groups of --OH, (C1 -C4) alkoxy, (C1 -C4) thioalkoxy, halogen, nitro, (C1 -C3) dialkylamino, or (C1 -C3) trialkylammonium-A- where A- is a pharmaceutically acceptable anion completing a salt;

Sp2 is a bond, --S--, or --O--, with the proviso that when Alk2 is a bond, Sp2 is also a bond;

Sp1 is a bond, --S--, --O--, --CONH--, --NHCO--, --NR'--, --N(CH2 CH2)2 N--, or --X--AR'--Y--(CH2)n --Z wherein X, Y, and Z are independently a bond, --NR'--, --S--, or --O--, with the proviso that when n=0, then at least one of Y and Z must be a bond and Ar' is 1,2-, 1,3-, or 1,4-phenylene optionally substituted with one, two, or three groups of (C1 -C5) alkyl, (C1 -C4) alkoxy, (C1 -C4) thioalkoxy, halogen, nitro, COOR', CONHR', O(CH2)n COOR', S(CH2)n COOR', O(CH2)n CONHR', or S(CH2)n CONHR', with the proviso that when Alk1 is a bond, Sp1 is a bond;

with the proviso that when Ar is unsubstituted 2,6-naphthylene or 1,3- or 1,4-phenylene optionally substituted with one group of (C1 -C6) alkyl or (C1 -C5) alkoxy and Alk2 is a bond, then Sp1 is not a bond, --O--, or --NHCO--.

FIG. 19A-B shows the amino acid sequences of the variable regions of the A33 light chain (FIG. 19A) and heavy chain (FIG. 19B). Sequences for the signal sequence are underlined and the mature variable region are shown in upper case. DNA sequences defined by the PCR primers are italicized. CDR regions are double underlined.

FIG. 20 shows oligonucleotide sequences used for the assembly of the A33 humanized light chain variable region. These oligonucleotide sequences are underlined. The expected coding sequence for the signal sequence is italicized, the mature variable region is in upper case, and the N terminal sequence of the human kappa constant region is in lower case, and all are shown below the underlined oligonucleotide sequences. The CDR regions and the non-CDR residues derived from the murine sequence are double underlined.

FIG. 21 shows oligonucleotide sequences used for the assembly of the A33 humanized heavy chain variable region. These oligonucleotide sequences are underlined. The expected coding sequence for the signal sequence is italicized, the mature variable region is in upper case, and the N terminal sequence of the human CH1 domain region is in lower case, and all are shown below the underlined oligonucleotide sequences. The CDR regions and the non-CDR residues derived from the murine sequence are double underlined.

FIG. 22 is a graph of a competition binding assay. Murine A33 and humanized A33 prepared from CHO-K1 transient expression experiments were used to compete for binding to Colo205 cells with FITC-labeled murine A33. Residual FITC-mA33 bound to cells was measured in a FACScan analyzer and fluorescence (Y axis) was related to input unlabeled antibody (X axis).

FIG. 23A-C are schematic diagrams of the chimeric and humanized A33 GS expression vectors. FIG. 23A is a schematic diagram of the chimeric A33(γ1) expression vector pGR50. FIG. 23B is a schematic diagram of the humanized A33(γ1) expression vector pAL71. FIG. 23C is a schematic diagram of the humanized A33 FAB'(γ4Δcys) expression vector pAL72. Only relevant restriction sites are shown.

Since the discovery of methodology for producing monoclonal antibodies was published in the 1970's (G. Kohler and C. Milstein, "Nature" 256, 495 (1975)), numerous attempts have been made to use these proteins to achieve selective targeting of antitumor agents to tumors. (E.g., see T. Ghose and A. H. Blair, "CRC Critical Rev. Drug Carrier Systems" 3, 263 (1987), G. A. Koppel, "Bioconjugate Chem." 1, 13 (1990), and J. Upeslacis and L. Hinman, "Ann. Rep. Med. Chem." 23, 151 (1988).) Although progress continues to be made in this field, most classical antitumor agents produce antibody conjugates which are relatively ineffective for a variety of reasons. Among the reasons for this ineffectiveness is the lack of potency of the chemo-therapeutic agent and its poor utilization due to the lack of efficient release of the drug at its site of action.

The potent family of antibacterial and antitumor agents, known collectively as the calicheamicins or the LL-E33288 complex, are described and claimed in U.S. Pat. No. 4,970,198 (1990). The most potent of the agents is designated γ1I, which is herein referred to simply as gamma. The dihydro derivatives of these compounds are described in U.S. Pat. No. 5,037,651 (1991) and the N-acylated derivatives are described in U.S. Pat. No. 5,079,233 (1992). Related compounds which are also useful in this invention include the esperamicins which are described and claimed in U.S. Pat. No. 4,675,187 (1987); 4,539,203; 4,554,162; and U.S. Pat. No. 4,837,206. All of these compounds contain a methyltrisulfide that can be reacted with appropriate thiols to form disulfides, at the same time introducing a functional group such as a hydrazide or similar nucleophile. Examples of this reaction with the calicheamicins are given in U.S. Pat. No.5,053,394 which also discloses targeted forms of the calicheamicins. All information in the above-mentioned patent citations is incorporated herein by reference. Two compounds which are useful for the synthesis of conjugates with carrier molecules, as disclosed and claimed in U.S. Pat. No. 5,053,394, are shown in Table 1.

Included as carrier molecules in U.S. Pat. No. 5,053,394 are steroids, growth factors, antibodies, antibody fragments, and their genetically or enzymatically engineered counterparts, hereinafter referred to singularly or as a group as carrier. The essential property of the carrier is its ability to recognize an antigen or receptor associated with an undesired cell line. Examples of carriers are given in U.S. Pat. No. 5,053,394, and such carriers are also appropriate in the present invention. Antibody carriers can be from almost any mammalian species (eg. mouse, human, dog, etc.) and can be produced by various methods (eg. murine antibodies via hybridomas, human antibodies via hybridomas from transgenetic mice, etc).

Specific examples of carriers which are exemplified herein are the antibodies P67.6, A33, CT-M-01 and the "anti-Tac" antibody of Waldman. These antibodies are used here in two forms: a murine form, designated by an "m" (e.g., m-P67.6), and a genetically engineered, humanized form, designated by an "h" (e.g., h-P67.6) whenever appropriate. The basic technology for humanization is disclosed by Winter in U.S. Pat. No. 5,225,539 (1993) and by Adair in WO 91/09967 (1991). m-P67.6 is disclosed in I. D. Bernstein et al., "J. Clin. Invest." 79, 1153 (1987) and recognizes the CD33 antigen which is prevalent on certain human myeloid tumors, especially acute non-lymphocytic leukemia (ANLL).

FIG. 1 and FIG. 2 show the DNA coding and predicted amino acid sequences of the variable regions of one particular h-P67.6 that is particularly suitable for use in the present invention. The framework for this antibody is the EU framework for human IgG4 shown in Gottlieb et al., "Biochemistry: 9, 3155 and 3161 (1970) and the amino acid sequences set forth in FIG. 1 (SEQ ID NO: 2) and FIG. 2 (SEQ ID NO: 4). The antibody was prepared using the general strategy described in WO 91/09967. It is first of all necessary to sequence the DNA coding for the heavy and light chain variable regions of the donor antibody, to determine their amino acid sequences. It is also necessary to choose appropriate acceptor heavy and light chain variable regions, of known amino acid sequence. The CDR-grafted chain is then designed starting from the basis of the acceptor sequence. It will be appreciated that in some cases the donor and acceptor amino acid residues may be identical at a particular position and thus no change of acceptor framework residue is required.

1. As a first step, donor residues are substituted for acceptor residues in the CDRs. For this purpose the CDRs are preferably defined as follows:

Heavy chain--CDR1: residues 26-35

--CDR2: residues 50-65

--CDR3: residues 95-102

Heavy chain --CDR1: residues 24-34

--CDR2: residues 50-56

--CDR3: residues 89-97 The positions at which donor residues are to be substituted for acceptor in the framework are then chosen as follows, first of all with respect to the heavy chain and subsequently with respect to the light chain.

2. Heavy Chain

2.1 Choose donor residues at all of positions 23, 24, 49, 71, 73 and 78 of the heavy chain or all of positions 23, 24 and 49 (71, 73 and 78 are always either all donor or all acceptor).

2.2 Check that the following have the same amino acid in donor and acceptor sequences, and if not preferably choose the donor: 2, 4, 6, 25, 36, 37, 39, 47, 48, 93, 94, 103, 104, 106 and 107.

2.3 To further optimize affinity, consider choosing donor residues at one, some or any of:

i. 1, 3

ii. 72, 76

iii. If 48 is different between donor and acceptor sequences, consider 69

3.3 To further optimize affinity, consider choosing donor residues at one, some or any of:

i. 1,3

ii. 63

iii. 60, if 60 and 54 are able to form potential salt bridge

iv. 70, if 70 and 24 are able to form potential salt bridge

v. 73, and 21 if 47 is different between donor and acceptor

vi. 37, and 45 if 47 is different between donor and acceptor

vii. 10, 12, 40, 80, 103,105

In order to transfer the binding site of an antibody into a different acceptor framework, a number of factors need to be considered.

1. The extent of the CDRs

The CDRs (Complementary Determining Regions) were defined by Wu and Kabat (Wu, T. T., and Kabat, E. A., J. Exp. Med. 132: 211-250 (1979); Kabat, E. A., et al., Sequences of Proteins of Immunological Interest, U.S. Dept. of Health and Human Services, NIH, 1987 (hereinafter "Kabat et al.") on the basis of an analysis of the variability of different regions of antibody variable regions. Three regions per domain were recognized. In the light chain, the sequences are 24-34, 50-56, 89-97 (numbering according to Kabat et al., Eu Index) inclusive and in the heavy chain the sequences are 31-35, 50-65 and 95-102 inclusive.

When antibody structures became available, it became apparent that these CDR regions corresponded in the main to loop regions which extended from the β barrel framework of the light and heavy variable domains. For H1 there was a discrepancy in that the loop was from 26 to 32 inclusive and for H2 the loop was 52 to 56 and for L2 from 50 to 53. However, with the exception of H1, the CDR regions encompassed the loop regions and extended into the β strand frameworks. In H1 residue, 26 tends to be a serine and 27 a phenylalanine or tyrosine, residue 29 is a phenylalanine in most cases. Residues 28 and 30 which are surface residues exposed to solvent might be involved in antigen-binding. A prudent definition of the H1 CDR therefore would include residues 26-35 to include both the loop region and the hypervariable residues 33-35.

It is of interest to note the example of Riechmann et al., Nature, 332: 323-3241 (1988), who used the residue 31-35 choice for CDR-H1. In order to produce efficient antigen binding, residue 27 also needed to be recruited from the donor (rat) antibody.

2. Non-CDR residues which contribute to antigen binding

By examination of available X-ray structures, a number of residues have been identified which may have an effect on net antigen binding and which can be demonstrated by experiment. These residues can be subdivided into a number of groups.

2.1 Surface residues near CDR (all numbering as in Kabat et al.).

2.1.1 Heavy Chain--Key residues are 23, 71 and 73. Other residues which may contribute to a lesser extent are 1, 3 and 76. Finally, 25 is usually conserved but the murine residue should be used if there is a difference.

2.1.2 Light Chain--Many residues close to the CDRs, e.g. 63, 65, 67 and 69 are conserved. If conserved, none of the surface residues in the light chain are likely to have a major effect. However, if the murine residue at these positions is unusual, then it would be of benefit to analyze the likely contribution more closely. Other residues which may also contribute to binding are 1 and 3, and also 60 and 70 if the residues at these positions and at 54 and 24, respectively, are potentially able to form a salt bridge, i.e., 60+54; 70+24.

2.2 Packing residues near the CDRs.

2.2.1 Heavy Chain--Key residues are 24, 49 and 78. Other key residues would be 36 if not a tryptophan, 94 if not an arginine, 104 and 106 if not glycines and 107 if not a threonine. Residues which may make a further contribution to stable packing of the heavy chain and hence improved affinity are 2, 4, 6, 38, 46, 67 and 69. 67 packs against the CDR residue 63 and this pair could be either both mouse or both human. Finally, residues which contribute to packing in this region but from a longer range are 18, 20, 80, 82 and 86. 82 packs against 67 and in turn 18 packs against 82. 80 packs against 69 and in turn 20 packs against 80. 86 forms an H bond network with 38 and 46. Many of the mouse-human differences appear minor, e.g., Leu-lle, but could have a minor impact on correct packing which could translate into altered positioning of the CDRs.

2.2.2 Light Chain--Key residues are 48, 58 and 71. Other key residues would be 6 if not glutamine, 35 if not tryptophan, 62 if not phenylalanine or tyrosine, 64, 66, 68, 99 and 101 if not glycines and 102 if not a threonine. Residues which make a further contribution are 2, 4, 37, 45 and 47. Finally, residues 73 and 21 and 19 may make long distance packing contributions of a minor nature.

2.3 Residues at the variable domain interface between heavy and light chains--In both the light and heavy chains most of the non-CDR interface residues are conserved. If a conserved residue is replaced by a residue of different character, is replaced by a residue of different character, e.g., size or charge, it should be considered for retention as the murine residue.

2.3.1 Heavy Chain--Residues which need to be considered are 37 if the residue is not a valine but is of larger side chain volume or has a charge or polarity. Other residues are 39 if not a glutamine, 45 if not a leucine, 47 if not a tryptophan, 91 if not a phenylalanine or tyrosine, 93 if not an alanine and 103 if not a tryptophan. Residue 89 is also at the interface but is not in a position where the side chain could be or great impact.

2.3.2 Light Chain--Residues which need to be considered are 36, if not a tyrosine, 38 if not a glutamine, 44 if not a proline, 46, 49 if not tyrosine, residue 85, residue 87 if not a tyrosine and 98 if not a phenylalanine.

2.4 Variable-Constant region interface--The elbow angle between variable and constant regions may be affected by alterations in packing of key residues in the variable region against the constant region which may affect the position of VL and VH with respect to one another. Therefore it is worth noting the residues likely to be in contact with the constant region. In the heavy chain the surface residues potentially in contact with the variable region are conserved between mouse and human antibodies, therefore the variable region contact residues may influence the V-C interaction. In the light chain, the amino acids found at a number of the constant region contact points vary, and the V & C regions are not in such close proximity as the heavy chain. Therefore the influences of the light chain V-C interface may be minor.

With reference to FIG. 1 and FIG. 2, the overlapping oligonucleotides that were synthesized (Oligo L1 through L8) are shown with double underlines, and the PCR assembly procedure (cf. WO 92/01059) was applied to these sequences. The CDR regions of the protein are designated with single underlines and other amino acids that were taken from the murine sequences are shown with a double underline. The restriction sites that were used to splice the sequences into plasmids are indicated at the beginning and end of the sequences. The variable portion of the heavy chain was cloned into plasmid pMRR14 (WO 93/06231) to give the plasmid designated pAL63 (FIG. 5) and the variable portion of the light chain was cloned into plasmid pMRR15 (FIG. 6) to give pAL60 (FIG. 7). Plasmids pMRR14 and pMRR15 contained the constant regions of the heavy and light chains, respectively, and therefore pAL63 and pAL60 contained complete sequences for the P67.6 heavy and light chains. Plasmid pMRR14 has an hCMV-MIE promoter, a polylinker site, and a nucleotide coding sequence which encodes the three constant domains of a human IgG4 antibody (FIG. 8). The plasmids were cotransfected into CHO-L761 cells to generate a h-P67.6 producing line from which the h-P67.6 was purified by standard methods. The resultant h-P67.6 bound to HL60 cells in competition with murine antibody with about a 50% loss in immunoaffinity. This binding was inhibited by pre-incubation with soluble CD33 antigen.

The antibody m-CT-M-01 is disclosed in E.P. 86 401482.4/0208615 and recognizes the polyepithelial mucin (PEM) antigen present on many human solid tumors, particularly breast, lung, and ovarian. The humanized version of this antibody, h-CT-M-01, is described in WO 93/06231 (1993). The procedures from WO 93/06231 which were employed to make h-CT-M-01 are as follows.

The DNA and predicted amino acid sequences for the unprocessed variable domains of the CTM01 heavy chain are shown in the Sequence Listing as SEQ ID NO: 7 and 8, respectively. The DNA and predicted amino acid sequences for the unprocessed variable domains of the CTM01 light chain are shown in the Sequence Listing as SEQ ID NO: 9 and 10, respectively. SEQ ID NO: 7 shows the sequence coding for the VH domain and SEQ ID NO: 8 shows the predicted amino acid sequence. The Leader Sequence for the heavy chain runs from residue 1 to residue 19 as shown in SEQ ID NO: 7. SEQ ID NO: 9 shows the sequence coding for the VL domain and SEQ ID NO: 10 shows the predicted amino acid sequence. The leader sequence for the light chain runs from residue 1 to residue 20 as shown in SEQ ID NO: 9. Examination of the derived amino acid sequences revealed considerable homology with other characterized immunoglobulin genes. The CTM01 MAb was confirmed to be an IgG1-kappa antibody.

Preparation of CDR-grafted Antibody Products

It was decided to use the EU human antibody framework (as defined by Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, U.S. Dept. of Health and Human Services, NIH, USA, 1987 and Wu, T. T. and Kabat, E. A., J. Exp. Med., 132: 211-250 (1970) (hereinafter "Kabat references")) for carrying out the CDR-grafting. The strategy followed for CDR-grafting was as set out in International Patent Specification No. WO-A-91/09967, which is described above.

Two CDR-grafted heavy chains were designed. In the first, gH1, all three CDRs (as defined by the Kabat references) were changed to murine residues. In addition, residues 2, 37, 71, 73, 94, 103, 104, 105 and 107, which are outside the Kabat CDRs, were also changed to murine residues. In the second gH2, in addition to those murine residues in gH1, residues 48, 67 and 69 were changed to murine residues with a view to improving packing of the VH domain.

Two CDR-grafted light chains were also designed. In the first, gL1, all three CDRs (as defined by the Kabat references) were changed to murine residues. In addition, residues 3, 36, 63 and 108, which are outside the Kabat CDRs, were changed to murine resides. In the second, gL2, in addition to those murine residues in gL1, residues 37, 45 and 48 were changed to murine residues with a view to improving packing.

A nucleotide sequence coding for the gH1 variable domain was produced by oligonucleotide assembly using oligonucleotides H1 to H8. The sequences for these oligonucleotides are given in the Sequence Listing under SEQ ID NOS: 11 to 18. The way in which these oligonucleotides are assembled to produce the gH1 coding sequence is shown in FIG. 9. The amino acid sequence coded for by this gH1 sequence is shown in the sequence listing under SEQ ID NO: 19.

A nucleotide sequence coding for the gH2 variable domain was also produced by oligonucleotide assembly using oligonucleotides H1, H2, H3A, H4, H5, H6A, H7 and H8. Oligonucleotide H3A differs from oligonucleotide H3 (SEQ ID NO: 13) in that residues 55 to 57 have been changed from GTG to GCA and residues 61 to 63 have been changed from ATT to CTG. Oligonucleotide H6A differs from oligonucleotide H6 (SEQ ID NO: 16) in that residues 70 to 72 have been changed from TAC to TAA. Thus, the gH2 variable domain encodes the same sequence as is shown under SEQ ID NO: 19, except that at residue 67, MET has been changed to ILE; at residue 87, VAL has been changed to ALA; and at residue 89, ILE has been changed to LEU.

A nucleotide sequence coding for the gL1 variable domain was produced by oligonucleotide assembly using oligonucleotides L1 to L8. The sequences for these oligonucleotides are given in the Sequence Listing under SEQ ID NOS: 20 to 27. The way in which these nucleotides are assembled is similar to that shown in FIG. 9 for the gH1 coding sequence (except that L is substituted for H). The amino acid sequence coded for by the assembled gL1 variable domain coding sequence is shown in the Sequence Listing under SEQ ID NO: 25.

A nucleotide sequence coding for the gL2 variable domain was produced by oligonucleotide assembly using oligonucleotides L1, L2A, L3A and L4 to L8. Oligonucleotide L2A differs from oligonucleotide L2 (SEQ ID NO: 21) in that residues 28 to 30 have been changed from CAG to GTA. Oligonucleotide L3A differs from oligonucleotide L3 (SEQ ID NO: 22) in that residues 25-27 have been changed from CAG to CTC, residues 49-52 have been changed from AAG to CAG and residues 59-61 have been changed from CAT to ATC. Thus, the gL2 variable domain encodes the same sequence as is shown under SEQ ID NO: 28, except that: at residue 23, Gln has been changed to Val; at residue 62, Gln has been changed to Leu; at residue 60, Lys has been changed to Gln; and at residue 73, Met has been changed to lle.

For gene assembly 1 pmol of H2-H8 or L2-L7 was mixed with 10 pmol H1 and H8 or L1 and L8 in a 100 ml reaction with 5U Taq polymerase. A PCR reaction was done using 30 cycles (95° C., 1 min.; 50° C. 1 min; 72° C. 1 min). The resulting fragments were cut with HindIII and Apal for VL with Bstb1 and SPII for VH.

The nucleotide sequences coding for gH1 and gH2 were cloned as HindIII-Apal fragments into plasmid pMRR014 (FIG. 8) to produce plasmids pAL51 and pAL52 (FIGS. 10 and 11, respectively).

The nucleotide sequences coding for gL1 and gL2 were cloned as HindIII-Apal fragments into plasmid pMRR010 (FIG. 12) to produce plasmids pAL47 and pAL48 (FIGS. 13 and 14, respectively).

Transient Expression of CDR-grafted/CDR-grafted Antibodies

The following pairs of plasmids: pAL47, pAL51; pAL47, pAL52; pAL48, pAL51; and pAL48, pAL52; were cotransfected into CHO-L761 cells.

Direct binding assays were carried out on the culture supernatants produced by the doubly transfected cell lines.

The results of these assays are shown in FIG. 15, together with some results for chimeric/CDR-grafted antibodies.

From all the direct binding assays referred to above, it can be determined that the order of binding activity of the various antibodies produced by transient expression is as follows:

cLcH3>gL1ch=gL1gH2>cLgH2=gL2H2=gL1gH1=gL2cH>gL2gH1.

(wherein:cL=chimeric light chain;

cH=chimeric heavy chain;

gL1=CDR-grafted light chain with lowest number of amino acid changes;

gL2=CDR-grafted light chain with highest number of amino acid changes;

gH1=CDR-grafted heavy chain with lowest number of amino acid changes;

gH2=CDR-grafted heavy chain with highest number of amino acid changes).

The more active variants (CLcH, gL1cH, gL1gH2 and gL2gH2) together with the CTM01 MAb were tested in a competition enzyme immunoassay (EIA). Microwell plates were coated with PEM obtained as follows:

An affinity column was prepared by attaching the CTM01 MAb to a suitable chromatographic medium in a conventional manner. In a first method, pooled human urine samples were applied directly to the affinity column. In a second method, human milk was subjected to low speed centrifugation to separate the cream from skimmed milk. The skimmed milk was then subjected to high speed centrifugation to product an aqueous and a iipid component. The aqueous component was applied to the affinity column.

Once the affinity column was loaded, by either of the two methods, column fractions were eluted at high and low pHs, neutralized and assayed for reactivity with the CTM01 MAb. Fractions showing reactivity were pooled and dialyzed. The pooled fractions contained the polymorphic epithelial mucin (PEM) recognized by the CTM01 MAb.

The CTM01 MAb was biotinylated and was used to compete with the four variants referred to above. Bound biotinylated CTM01 MAb was revealed and quantified using a streptavidin-HRP conjugate and TMB.

The results of the competition EIA are shown in FIG. 16, which shows the same ranking of binding activity as set out above, except that the gL1cH combination shows greater activity than the cLcH combination.

It can thus be seen that CDR-grafted antibodies which recognize the same antigen as the CTM01 MAb have successfully been produced. The antibody m-A33 is disclosed in U.S. Pat. Nos. 5,160,723 and 5,431,897 and is a murine antibody which recognizes a glycoprotein antigen present on colon cancer cells. The humanized version of this antibody, h-A33, is disclosed in UK Patent Application 9,315,249.4. Examples of human frameworks which may be used to construct CDR-grafted humanized antibody molecules (HAMs) are LAY, POM, TUR, TEI, KOL, NEWM, REI and EU (Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, U.S. Dept. of Health and Human Services, NIH, 1987) KOL and NEWM are suitable for heavy chain construction. REI is suitable for light chain construction and EU is suitable for both heavy chain and light chain construction. Preferably, however, the LAY framework is used as the human framework for both heavy and light chain variable domains in view of its high level of homology with MAb A33.

To demonstrate antigen binding both direct and competition ELISA format binding assays were used using solid phase anti-Fcg chain to capture the antibody. In the direct binding assay ASPC-1 or Colo205 cells were incubated at 4-- C for 1 hour in the presence of various amounts of murine or humanized A33, or non-specific antibody controls. After washing the cells to remove unbound antibody, the presence of bound antibody was revealed by further incubation with FITC-labelled anti-murine or anti-human Fc and by detection in the FACScan analyzer (Becton Dickinson).

In competition format increasing amounts of the test antibody were coincubated with saturating amounts of FITC-labelled murine antibody and the ASPC-1 or Colo205 cells as above. After washing the cells to remove unbound antibody, the binding of the FITC labelled murine antibody to the cells was detected in the FACScan analyzer.

Cloning of A33 Variable Region Sequences

Murine A33 (IgG2a/k), (Welt, S., et al., J. Clin. Oncol. 8: 1894-1896 (1990)), was obtained from culture of 1.5 L of hybridoma supernatant. (ATCC, HB 8779). 32.5 mg was purified by Protein A Sepharose. This material was used as an assay standard and the separated heavy and light chains were subjected to N terminal sequencing.

The PCR amplified products were cleaved with BstBI and SpII for the light chain and HindIII and Apal for the heavy chain. These fragments were cloned into the human kappa light chain acceptor vector, pMRR15.1, and the human heavy chain, IgG1, acceptor vector, pMRR011, respectively, to give chimeric expression vectors pRO108 for the light chain (FIG. 18) and pRO107 for the heavy chain (FIG. 18), respectively.

For each plasmid the variable regions from four independent clones were sequenced. For both variable regions the DNA sequence between the priming regions was the same in each of the four clones. Within the priming region sequence variability was seen, derived from the redundancy in the sequences of the primers used. For both heavy and light chains, the deduced amino acid sequences obtained for the first 11 residues of the mature variable domain were in agreement with the results of N terminal peptide sequencing of the murine antibody.

The DNA sequences were further confirmed by a second PCR experiment using forward primers that anneal in framework 1 (Orlandi, R., et al., Proc. Natl. Acad. Sci. USA 86:3833-3837 (1989)). The sequences obtained (SEQ ID NOS: 54 and 56) agreed with those found in the first experiment. The amino acid sequences of the A33 light and heavy chain variable regions are shown in FIG. 19 and SEQ ID NOS: 55 and 57.

Design of Humanized A33

The murine variable regions of A33 were humanized according to the strategy described in Adair et al., (1991), and by reference to other recently published data on antibody humanization (Co, M. S., et al., Proc. Natl. Acad. Sci. USA 88:2869-2873 (1991)). The VH of A33 shows closest homology (70%) to the consensus sequence of human subgroup VH III, while the VL shows greatest homology to the consensus sequence of VL I and VL IV (62%). From these subgroups LAY, which has a VH III heavy chain and VL I light chain, was chosen as the human framework. For the light chain residues 1-23, 35-45, 47-49, 57-86, 88 and 98-108 inclusive were derived from the LAY sequence, (numbering as in Kabat et al., 1987) and the residues 24-34, 46, 50-56, 87 and 89-97 inclusive were derived from the murine sequence. Residues 24-34, 50-56 and 89-97 correspond to the Complementarity Determining Regions (Kabat et al., 1987)(see FIG. 20). Residues 46 and 87 are predicted to be at the interface of the light and heavy variable regions. Residue 46 is usually a leucine. Residue 87 is usually either a phenylalanine or tyrosine.

For the heavy chain residues 2-26, 36-49, 66-71, 74-82a, 82c-85, 87-93 and 103 to 113 inclusive were derived from the LAY sequence while residues 1, 27-35, 50-65, 72, 73, 82b, 86 and 94-102 inclusive were derived from the murine sequence (see FIG. 21). Residues 31-35, 50-65 and 95-102 in the heavy chain correspond to the Complementarity Determining Regions (Kabat et al., 1987). The murine derived amino acids in the framework regions were included for the following reasons. Residue 1 is usually solvent accessible and in the vicinity of the CDR region. LAY has a residue, alanine, not normally found at this position in human or murine VH sequences and therefore the murine residue was used. At positions 72 and 73 the murine residue was used because of the predicted proximity to CDR2 and also, in the case of residue 72, to remove the possibility of introducing an N-linked glycosylation site into the variable domain by the use of the LAY framework (see also Co, M. S., et al., Proc. Natl. Acad. Sci. USA 88:2869-2873 (1991); European Patent Application No. 0438310 ("Law et al., 1991")). The murine sequence was also used at the interdomain residue 94, where A33 has a proline, not normally found at this position. Murine residues were used at positions 82b and 86 because the use of the human amino acids at these positions in a humanized antibody with LAY frameworks have previously been found to be deleterious for the expression of the heavy chain (International Patent Specification No. WO 92/010509).

Construction of Humanized A33 and Expression in Cho Transients

The humanized variable regions were assembled from overlapping oligonucleotides using a PCR assembly procedure (International Patent Specifications Nos WO 92/010509 and WO 92/011383, also Daugherty, B. L., et al., Nucl. Acids Res. 19:2471-2476 (1991); Law et al. 1991). The oligonucleotides are given in FIG. 20 (SEQ ID NOS: 58 to 65) for the light chain and FIG. 21 (SEQ ID NOS: 66 to 73) for the heavy chain. The oligonucleotides were assembled using either 1 pmole of the longer internal oligonucleotides for the heavy chain variable region or 0.01 pmole for the light chain, with 10 pmole of the shorter terminal oligonucleotides in both cases. The reaction conditions were 30 cycles of 92° C., 1 minute; 55° C. 1 minute; 72° C. 1 minute using Taq polymerase. The PCR products were digested with the appropriate restriction enzymes as described for the chimeric antibody constructions and cloned into pMRRO15.1 for the light chain, and pMRR011 for the heavy chain to give pCG16 and pRO109 respectively.

pRO 108 (CL expression vector) and pCG 16 (hL expression vector) were each co-transfected with pRO107 (cH expression vector) or pRO109 (hH expression vector) into CHO L761h cells, and the antibody in the culture supernatant was calibrated and shown to compete for binding with FITC-labelled murine A33 for antigen on Colo205 cells by FACScan analysis (FIG. 22). The relative potency of the fully humanized antibody was calculated to be 75% of that of the murine antibody based on the competition IC50 values.

Construction of Stable Cell Lines in NSO for hA33(γ1), hFab'(γ4Δcys)

Expression vectors based on the GS amplification system (Bebbington, C. R., et al., Bio/Technology 10:169-175 (1992); European Patent Specification No. 256055) were constructed for the humanized A33 and also for a vector capable of producing a humanized Fab'(γ4Δcys) fragment.

The humanized A33 expression vector pAL71 (FIG. 23), capable of coexpressing both hL and hH chains, was constructed by obtaining a Not1-BamH1 vector fragment from pCG16 which contains the shuttle vector sequences, the GS cDNA selectable marker, a copy of the hCMV-MIE promoter/enhancer 5' to the humanized light chain which is followed by the SV40 polyadenylation sequences and the Ig terminator sequence. This vector fragment was combined with the Not1-BamH1 fragment from pRO109 which contains the humanized A33 (γ1) heavy chain between the hCMV/MIE promoter/enhancer and the SV40 polyadenylation sequences.

The heavy chain fragment which combines with light chain to give an antibody Fab' fragment consists of the heavy chain variable domain, the CH1 domain and the hinge sequence (or derivative hinge sequence) and is known as the Fd' fragment. A modified Fd' sequence, in which the hinge sequence has been altered to substitute one of the cysteines for alanine, and so reduce the number of hinge cysteines to one is described in Bodmer, M. W., et al., International Patent Specification No. WO 89/01974 (1989) and is known as the Fd'(γ4ΔCys) sequence.

An expression vector capable of producing A33 hL and A33 hH-Fd'(γ4ΔCys), pAL72 (FIG. 23), was constructed by combining the Not1-BamH1 vector fragment from pCG16 with the Not1-Apa1 fragment from pRO109, which encodes the hCMV-MIE promoter/enhancer 5' to the humanized A33 heavy chain variable region, along with IgG4 CH1 and hinge(DCys) sequences and SV40 polyadenylation sequences on an Apa1-BamH1 fragment derived from the expression vector pAL49 (International Patent Specification No. WO 92/010509).

Plasmids pGR50, pAL71, and pAL72 were linearized with Pvu1 for pGR50, or Fsp1 for pAL71 and pAL72, and 50 mg of DNA was used to transfect 107 NSO cells, by electroporation, using 1500 V and 3 mF with 2×1 second pulses. The cells were distributed into 96 well dishes at 5×105 cells in 50 mL per well in CB2 medium supplemented with 10% dialyzed, heat inactivated, fetal calf serum (10% of dFCS) and 2mM glutamine. After 24 hours at 37° C., 5% CO2 a further 100 mL of CB2 medium containing 10% dFCS, supplemented with 10 mM methionine sulphoximine (MSX) was added to each well. The cells were incubated for 2-3 weeks. Discrete colonies were observed after 19 days of culture. Culture supernatants were harvested from wells containing single colonies and antibody producing colonies were expanded for estimation of specific production rates (SPR) as picograms (pg) of antibody produced per cell, per 24 hours of culture. The cell lines with the highest SPR values were taken for further analysis. Cell stocks were frozen and the cell lines were grown in by transfer into MM1 medium containing<1% dialyzed fetal calf serum, to produce culture supernatants for antibody purification. Some loss of productivity was seen initially with the clgG1 and hlgG1 cell lines but after culture in MM1 the productivity of the cell lines stabilized.

Anti-Tac is disclosed in T. A. Waldman et al., "J. Immunol." 126, 1393 (1981) and is a murine antibody reactive with the IL-2 receptor that is found on activated and functionally mature T cells, including abnormally activated leukemia cells. The procedure for making the Anti-Tac antibody described by T. A. Waldman et al. is as follows:

Production of Anti-Tac Monoclonal Antibody

Cells used for immunization. Cells used for immunization were human cultured T cells (Called CTC 16) that were derived from peripheral blood T cells from a patient with mycosis fungoides and that were continuously growing in the presence of T cell growth-promoting factors contained in the conditioned medium from phytohemagglutinin(PHA)-stimulated lymphocyte cultures (Uchiyama, T., et al., "Immunoregulatory functions of cultured human T lymphocytes," (1980) Trans. Assoc. Am. Phys.; Morgan, D. A., et al., (1976) Science 193:1007). CTC 16 had been in culture for 172 days when they were injected into mice. Cell surface marker analysis showed that more than 95% of these CTC formed spontaneous rosettes with sheep erythrocytes and reacted with the heteroantiserum to la (anti P23, Sarmiento, M., et al., (1980) Proc. Natl. Acad. Sci. 77:1111).

Screening for reactivity of hybridoma culture supernatants. Fifteen days after cell fusion, the culture supernatants from wells with cell growth were tested for their antibody activity by a complement-dependent cytotoxicity test. CTC 16 and an Epstein-Barr virus (EBV) transformed B cell line (16B), both of which were derived from the same patient, were labeled with 51 Cr sodium chromate (Amersham Searle, Arlington Heights, Ill.), and were used as target cells. Fifty μl of hybridoma culture supernatants and 50 μl of target cells at the concentration of 5×105 /ml were mixed in round-bottom microtiter plates (Linbro Scientific Inc., Hamden, Conn.) and incubated at 37° C. for 30 min; then 25 μl of appropriately diluted neonatal rabbit serum as a complement source was added. After another 60 minute incubation at 37° C., the supernatants were collected by a Titertek supernatant Collecting System (Flow Laboratory Inc., Rockville, Md.) and counted with a gamma counter. Specific cytotoxicity was calculated as follows: ##EQU1##

Cloning and production of a hybridoma antibody. After screening, hybridoma cultures that showed cytotoxic activity against CTC 16 but not 16B were expanded and cloned by a limiting dilution method. Irradiated (2000 R) rat fibroblasts (Flow Laboratory, McLean, Va.) were used as a feeder layer. Fourteen days later, when hybridoma clones had been expanded, the supernatants were tested again for their cytotoxic activity against the same target cells, using the 51 Cr release cytotoxicity test, and 1 clone-producing antibody reactive with CTC 16 but not 16B was selected and expanded. These cloned hybridoma cells were cultured in 250-ml plastic culture flasks. Supernatants were collected when the cell concentration was between 5×105 and 9×105 /ml. Culture supernatants obtained on a particular day were used in these studies. In order to obtain ascites containing large quantities of antibodies, 2×105 hybridoma cells were injected i.p. into BALB/c mice primed with pristane (Aldrich Chemical Co., Milwaukee, Wis.). The monoclonal antibody studied was demonstrated to be of the mouse IgG2a subclass by the positive immunoprecipitation reaction in agarose of ascites fluid with goat anti-mouse IgG2a.

The two basic types of conjugates disclosed in U.S. Pat. No. 5,053,394 are those which are attached to lysine residues of the antibody and those which are attached to the oxidized carbohydrate residues using the method taught in U.S. Pat. No. 4,671,958. Lysine attachment as it is disclosed in U.S. Pat No. 5,053,394 produces conjugates which are stable to hydrolysis under normal physiological conditions. The carbohydrate-based conjugates, which involve the formation of a hydrazone from a hydrazide or similar derivative, are hydrolytically unstable under certain conditions, and that is in many cases an advantage. Some instability is often needed to allow release of the drug once the conjugate has been internalized into the target cell, but a certain degree of stability is important to prevent premature release of the drug from the antibody. However, these carbohydrate-based conjugates suffer from various drawbacks. First, it is necessary to use periodate to generate aldehydes from the carbohydrate residues of the antibody. Antibodies contain cysteines, cystines, methionines, tryptophans, or tyrosines residues which are necessary for proper functioning of the antibody. However, these same amino acids can be sensitive to periodate oxidation, and if such oxidation takes place to an amino acid which either is part of the antigen binding site of the antibody or a structurally important region near the antigen binding site, its immunoaffinity can be significantly diminished. A second drawback of using the carbohydrates for conjugation is the variability of the hydrazones and related structures that are generated from the naturally-occurring sugars and the hydrazide derivative. Not only are the hydrazones subject to different rates of hydrolysis due to differences in their local structure, but other structures, such as hydrated species, piperadines, etc. can also be generated. Any one conjugate may contain structures that are either too stable or too labile for optimum activity.

Limited examples of how to combine some of the properties of the carbohydrate-based conjugates and the lysine-based conjugates have appeared using other less potent classes of anticancer agents. Cullinan in U.S. Pat. No. 5,006,652 and 5,094,849 teaches that certain bifunctional compounds containing both carboxylic acid and aldehyde or keto functionality can be used as spacers between the lysines of antibodies and hydrazide derivatives of the Vinca alkaloids, while Johnson in U.S. Pat. No. 5,028,697 and 5,144,012 teaches similar art for methotrexate analogs. Sinam et al. also disclose similar constructs in WO Pat. No. 90/03401. In none of these cases is it demonstrated that this method is useful for preparing conjugates of the methyltrisulfide antitumor antibiotics, especially the calicheamicins or esperamicins. The cited patents do not demonstrate that these constructs made with either the Vinca alkaloids, the methotrexate analogs, or other agents are superior in their biological profile to conjugates made using lysine-based or carbohydrate-based conjugates.

The present invention describes a series of conjugates prepared from the potent methyltrisulfide antitumor antibiotics made with an improved linker system that gives conjugates which in many cases are vastly superior biologically to conjugates of the same drugs made by other methods.

DETAILED DESCRIPTION OF THE INVENTION

The conjugates of this invention use linkers that can be added to a derivative of a drug, particularly hydrazides and related nucleophiles, prepared from the methyltrisulfide containing antitumor antibiotics. The linkers require a carbonyl group on one end for formation of a Schiff's base, particularly a hydrazone, and a carboxylic acid on the other end. The carboxylic acid can be activated and subsequently reacted with the lysines of an antibody or other targeting protein or with an amine, alcohol, or other appropriate nucleophile on other targeting agents which have been chosen for their ability to target undesired cell populations. These constructs, which for antibodies contain elements of both the lysine-based conjugates and the carbohydrate-based conjugates, not only overcome the disadvantages of previously disclosed constructs, but have the additional advantage that they can be fine-tuned by varying the structure of the linker to "design in" the optimum amount of hydrolytic stability/instability. This can result in maximum toxicity to the target cells with minimal toxicity to the non-target cells. The optimum hydrazone stability/instability is not necessarily the same for each drug and targeting agent combination.

The method of constructing the conjugates described in this patent produces conjugates of the methyltrisulfide antitumor antibiotics which are unexpectedly stable relative to the carbohydrate based conjugates without loss of activity. In some cases, the conjugates are 100 times more potent than the corresponding conjugates made by the carbohydrate-based method and, in addition, show reduced cytotoxicity against non-target cell lines. This results in conjugates with up to 10,000-fold selectivity between target and non-target cell lines.

The linkers required for the construction of these conjugates can be represented by the following formula:

Z3 CO-Alk1 -Sp1 -Ar-Sp2 -Alk2 -C(Z1)=Z2 !m

Alk1 and Alk2 are independently a bond or branched or unbranched (C1 -C10) alkylene chain. Sp1 is a bond, --S--, --O--, --CONH--,--NHCO--, --NR'--, --N(CH2 CH2)2 N--, or --X--AR'--Y--(CH2)n --Z wherein n is an integer from 0 to 5, X, Y, and Z are independently a bond, --NR'--, --S--, or --O--, and AR' is 1,2-, 1,3-, or 1,4-phenylene optionally substituted with one, two, or three groups of (C1 -C5) alkyl, (C1 -C4) alkoxy, (C1 -C4) thioalkoxy, halogen, nitro, COOR', CONHR', O(CH2)n COOR', S(CH2)n COOR', O(CH2)n CONHR', or S(CH2)n CONHR' wherein n is as hereinbefore defined, with the proviso that when Alk1 is a bond, Sp1 is also a bond. R' is a branched or unbranched (C1 -C5) chain optionally substituted by one or two groups of --OH, (C1 -C4) alkoxy, (C1 -C4) thioalkoxy, halogen, nitro, (C1 -C3) dialkylamino, or (C1 -C3) trialkylammonium--A- where A- is a pharmaceutically acceptable anion completing a salt. Sp2 is a bond, --S--, or --O--, with the proviso that when Alk2 is a bond, Sp2 is also a bond. Z3 is a hydroxyl group, and m is 1.

The groups Alk1, Sp1, Alk2 and Sp2 in combination, as well as the group Ar discussed below, allow for spacing of the carbonyl group from the carboxylic acid. Furthermore, Alk1 and Sp1 can influence the reactivity of the carboxyl group both during and after it has been activated. When Alk2 and Sp2 together are a bond, the Sp1 group also influences the reactivity of the carbonyl group on the other end of the linker and the stability of the product formed from reactions at that carbonyl. The group R' can be used to influence the solubility and other physiochemical properties of these compounds. A preferred embodiment for Alk1 is (C2 -C5) alkylene, and for Sp1 is an oxygen atom. A preferred embodiment for the groups Alk2 and Sp2 together is a bond.

With reference to the structure shown above, the group z2 is an oxygen atom. The group Z1 is H, (C1 -C5) alkyl, or phenyl optionally substituted with one, two, or three groups of (C1 -C5) alkyl, (C1 -C4) alkoxy, (C1 -C4) thioalkoxy, halogen, nitro, COOR', CONHR', O(CH2)n COOR', S(CH2)n COOR', O(CH2)n CONHR', or S(CH2)n CONHR', wherein n and R' are as hereinbefore defined. The group Z1 has a pronounced effect on the reactivity of the carbonyl group and on the stability of the products formed from reactions at the carbonyl. When Z1 is aryl and the product is, for example, a hydrazone, the hydrazone is relatively stable; when Z1 is hydrogen, then an intermediate level of stability is obtained, and when Z1 is (C1 -C6) alkyl, relatively less stable hydrazones are formed. As stated earlier, stability is important to prevent premature release of the drug from the antibody, but some instability is needed to allow release of the drug once the conjugate has been internalized into target cells. A preferred embodiment for the Z1 group is (C1 to C3).

The choice of Ar has a significant influence on the stability of the products derived from the carbonyl when Alk2 and Sp2 are together a bond. Both the relative position of Sp1 and Sp2 as well as the presence of additional substituents on Ar can be used to fine-tune the hydrolytic behavior of the product formed from the carbonyl. A preferred embodiment for Ar is 1,2-, 1,3-, or 1,4-phenylene, or 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-, 1,8-, 2,3-, 2,6-, or 2,7-naphthylidene.

The structures of specific examples of linkers which are useful in the present invention are as follows: ##STR9##

Only a few of the more simple of these linkers are commercially available, i.e., linkers 1, 2, 3, 19, 23, 24, and 33. Linker 20 is listed by the Chemical Abstract Services with registry number 5084-45-7. Many linkers which contain aryl ethers as a part of their structure, such as 7, 8, 10, 13, 14, 15, 16, 17, 20, 21, 25, 28, 30, and 31, can be made by alkylating a phenolic ketone with an electrophile, such as ethyl 4-bromobutyrate, using an appropriate base, such as potassium carbonate, in an appropriate solvent, such as N,N-dimethyl formamide, and then converting the ester into the required carboxylic acid by hydrolysis with, for example, sodium hydroxide or potassium carbonate in aqueous methanol. This strategy can also be used with linkers such as 5, 6, 9, 11, 18, or 27, where the carbonyl is carried through the initial steps of the preparation in a masked form, such as an olefin or an alcohol. The carbonyl can then be generated later, as described in the examples, by oxidation with ozone or pyridinium chlorochromate, resp. This procedure is especially valuable when a more reactive carbonyl is present in the final linker.

When necessary, the required carboxylic acid can be introduced in a masked form as in the preparation of linker 26. In this case the phenol is alkylated with 5-bromo-1-pentene and the acid is liberated from the olefin by reaction with ozone followed by pyridinium chlorochromate oxidation. Linkers such as 22 or 32 can be made by alkylating an appropriate secondary amine (a piperazine or phenothiazine derivative, resp.) with an appropriate electrophile and then exposing the required carboxylic acid in a later step, similar to the previously mentioned strategies. Linker 12 was made by reduction of the corresponding cinnamate with hydrogen. Although this reaction gave a relatively impure product, the crude mixture was useful for conversion to the required hydrazone because none of the by-products contained aldehyde groups. Structures with more elaborate substituents, such as linkers 33, 34, 35, or 36, can be made from simpler structures by, for example, reacting an ester with an appropriate nucleophile or by quaternizing an amine with an electrophile, such as methyl iodide.

The linkers defined above can be used to form conjugates as follows: ##STR10## With reference to Scheme 1 above, the linker of structure A, wherein Z1, Alk1, Sp1, Ar, Sp2, and Alk2 are as hereinbefore defined, is condensed with a compound of structure W-S-S-Sp-Q, which itself is derived from a methyltrithio antitumor antibiotic, and wherein W is ##STR11##

The condensation can be run in most compatible organic solvents, but is particularly efficient in alcoholic solvents such as methanol or ethanol. This condensation reaction is acid catalyzed. The carboxylic acid in the linkers themselves is sufficient in many cases to catalyze this reaction, but adding a compatible acid catalyst, such as about 5% acetic acid, helps improve the rate of reaction in many cases. The temperature of this reaction can be from about ambient temperature to the reflux temperature of the solvent. The products are isolated in pure form by removing the volatile solvents and purifying the mixture by chromatography on a suitable medium such as BIOSIL A™, a modified silica gel available from Bio-Rad. It should be understood that the products of structure B, as well as the products from the further transformation of these compounds, exist as easily-interconverted syn and anti isomers at the locus defined as Q, and that these products can exist in different hydrated forms, depending on the exact conditions of solvent and the pH at which these compounds are examined. Such differing physical forms are also included within the scope of this patent.

The carboxylic acid of structure B (Z3 =--OH) is next converted to an activated ester in preparation for conjugation of these intermediates with carrier molecules. Such transformations convert Z3 (structure B) to halogen, --N3, ##STR12##

For example, reaction of the carboxyl form of structure B (Z3 =--OH) with a coupling agent, such as 1,3-dicyclohexylcarbodiimide or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide or other comparable carboxyl-activating group in an inert solvent, such as N,N-dimethylformamide, tetrahydrofuran, or acetonitrile, leads to the formation of an activated ester, such as the N-hydroxysuccinimide ester described herein. These active esters can be isolated in pure form by removal of the volatile solvents and chromatography on an appropriate medium, such as BIOSIL A™. Alternately, the coupling reaction can be quenched with a polymeric carboxylic acid, filtered, and stripped of organic solvents, and the crude product can be used in the following step without further purification. This is especially useful if the active ester is difficult to handle, such as when ##STR13##

The final step in the construction of the conjugates of this patent involves the reaction of an activated ester (structure C) with a targeting molecule, as shown in Scheme 1. This produces a compound of structure D, wherein Z1, Z2, Alk1, Sp1, Ar, Sp2, and Alk2 are as hereinbefore defined, m is 0.1 to 15, and Z3 is a protein such as a growth factor or a mono- or polyclonal antibody, their antigen-recognizing fragments, or their chemically or genetically manipulated counterparts or a steroid, wherein a covalent bond to a protein is an amide formed from reaction with lysine side chains and the covalent bond to a steroid is an amide or an ester.

This conjugation reaction can be carried out in various appropriate buffers, such as borate, phosphate, or HEPES at slightly basic pH (pH˜7.4 to 8.5). The final construct can then be purified by appropriate methods, such as gel-exclusion chromatography, to remove unattached drug and aggregates to yield monomeric conjugates. This sequence of steps constitutes Method A as described in greater detail in the Examples section of this patent.

Alternative methods for constructing the conjugates of Scheme 1 are also contemplated as shown in Scheme 2. ##STR14##

For example, the linker (structure A as defined above) can be converted to an active ester and reacted with the targeting molecule prior to the reaction with the drug. Such manipulations convert structure A into structure E, wherein Z1, Alk1, Sp1, Ar, Sp2, and Alk2 are as hereinbefore defined, Z2 is an oxygen atom, and Z3 is halogen, --N3, ##STR15##

The activated ester is then reacted with the carrier to produce structure F, wherein Z1, Alk1, Sp1, Ar, Sp2, and Alk2 are as hereinbefore defined, Z2 is an oxygen atom, m is about 1 to about 20, and Z3 is a protein selected from mono- and polyclonal antibodies, their antigen-recognizing fragments, and their chemically or genetically manipulated counterparts and growth factors and their chemically or genetically manipulated counterparts, wherein a covalent bond to the protein is an amide formed from reaction with lysine side chains, or a steroid, wherein the covalent bond to the steroid is an amide or an ester.

Once the targeting molecule has been modified with the linker, it can be reacted with a compound of structure Q-Sp-S-S-W, which itself is derived from a methyltrithio antitumor antibiotic, and wherein W and Sp are as hereinbefore defined, and Q is H2 NHNCO--, H2 NHNCS--, H2 NHNCONH--, H2 NHNCSNH--, or H2 NO-- to produce a compound of Structure D (vida supra).

This sequence of steps in Scheme 2 constitutes Method B in the Examples section of this patent. Similar antibody-carbonyl constructs are covered in U.S. Pat. No. 5,144,012 mentioned above. Most of the linkers exemplified herein are new and offer the advantage that a much broader range of structural types and hence a broader range of stabilities is demonstrated. As a specific example, the acetophenone linkers, which are new to this patent, produced conjugates with better hydrolytic release properties of drug and which are more potent when used with the examples of the antibodies shown here. Specifically, the two conjugates prepared from h-P67.6 using 4-formylbenzenepropanoic acid or 4-acetylbenzenebutanoic acid condensed with calicheamicin N-acetyl gamma hydrazide (the two conjugates only differ by having Z1 =--H and Z1 =--CH3, respectively, in structure 3 of FIG. 24) gave in vitro IC50 's of 1.0 and 0.012 ng/mL, and specificity indices of 950 and 26,000, respectively. Although the acetophenone based linkers are seen to be superior in this case, it is not necessarily easy to predict which linker will be superior for any given targeting agent-drug construct.

BIOLOGICAL CHARACTERIZATION

Assessment of the biological properties of the conjugates included measuring their ability to recognize the antigen on target cell lines, relative to the unmodified antibody, and determining their selectivity and cytotoxic potentials, using the following methods:

IMMUNOAFFINITY ASSAYS

Relative immunoaffinities of conjugates are determined in a competitive binding assay in which varying concentrations of test conjugate are allowed to compete with a fixed amount of the same antibody labeled with 125 I-Bolton Hunter reagent for binding to a fixed number of cells. For m- or h-P67.6, HEL 92.1.7 human erythroleukemia cells ATCC (American Type Culture Collection) TIB 180! are used at a concentration of 107 cells/mL; for CT-M-01, cell line A2780DDP (E. M. Newman, et al., "Biochem. Pharmacol." 37, 443 (1988)) is used; and for m- or h-A33, cell line COLO 205 (ATCC CCL 222) is used. The concentration of test conjugate required to obtain 50% inhibition of binding of the labeled antibody to target cells is compared with the concentration of a reference preparation of native antibody required for 50% inhibition.

Samples for assay are adjusted to ˜300 μg protein/mL in medium and six serial four-fold dilutions of each are prepared in medium (RPMI-1640 containing 5% heat-inactivated fetal calf serum), for a total of seven concentrations of each sample. The reference antibody is diluted in the same way. An aliquot of 0.05 mL of each dilution is transferred to a 12×75 mm plastic tube, and 0.05 mL of labeled reference antibody at 4 μg/mL is added. The tubes are mixed and chilled at 4° C. Then 0.1 mL of chilled cell suspension is added to each tube. All tubes are mixed again, and incubated for 1 hr at 40° C.

Controls to determine maximal binding and non-specific binding are included in each assay. Maximal binding is determined by mixing 0.05 mL of medium, 0.05 mL of 125 I-antibody, and 0.1 mL of cells; non-specific binding is determined by mixing 0.05 mL of 500 μg/mL of native antibody, 0.05 mL of iodinated antibody, and 0.1 mL of cells.

At the end of the incubation, cells are washed twice, by centrifugation and resuspension, with 3 mL of cold PBS each time. The cells are resuspended in 0.5 mL of PBS, transferred to clean tubes, and radioactivity is determined in a gamma-counter.

The percent inhibition of binding is calculated by the following equation: ##EQU2## The percent inhibition values are plotted against sample concentrations, and from the resulting curves the sample concentration that gives 50% inhibition of binding (IC50) is interpolated. The relative immunoaffinity of each tested conjugate is then determined as follows:

Relative Immunoaffinity=IC50 (reference)÷IC50 (sample)

IN VITRO CYTOTOXICITY ASSAY

Cytotoxic activities are determined in an in vitro pulse assay in which varying concentrations of test conjugate are incubated with antigen-positive and antigen-negative cells for 1 hr, then cultured for three days. Viability is assessed by 3 H!thymidine incorporation during the final 24 hr of culture. As a measure of potency, the concentration of test conjugate required to inhibit 3 H!thymidine incorporation by 50% (IC50) is determined from the titration curve. The specificity is determined by comparing IC50 values on antigen-positive and antigen-negative cells for P67.6, A33, and m-CT-M-01 or by use of a conjugate of the same drug with the non-targeting antibody P67.6 for h-CT-M-01 conjugates or MOPC-21 for anti-Tac conjugates. MOPC-21 (F. Melchers, "Biochem. J." 119, 765 (1970)) is an antibody which does not recognize any normally occurring, physiologically pertinent antigen.

Samples for assay are readjusted to ˜1 μg/mL of drug equivalents in medium and five serial ten-fold dilutions of each are prepared in medium, for a total of six concentrations of each sample. In addition, a medium control is included with each sample set, as well as calicheamicin N-acetyl gamma as a drug control. An aliquot of 0.1 mL of cell suspension is added to 17×100 mm plastic tubes containing 0.1 mL of sample; a separate series of tubes is prepared for each cell line. The tubes are loosely capped and incubated for 1 hr at 37° C. in a humidified atmosphere of 5% CO2 in air. At the end of the incubation, cells are washed twice by centrifugation and resuspended with 8 mL of medium each time. Cell pellets are resuspended in 1 mL of medium and plated in triplicate in 96-well microtiter plates at 0.2 mL/well. The plates are incubated for 2 days at 37° C. as above. Then 0.1 mL of medium is removed from each well and replaced with 0.1 mL of fresh medium containing 0.1 μCi of 3 H!thymidine. Plates are returned to the incubator for one more day. Plates are frozen and thawed, and cells are harvested on glass fiber filter mats. The amount of 3 H!thymidine incorporated is determined by liquid scintillation counting.

The measured cpm of the triplicate cultures of each sample dilution are averaged and the percent inhibition of 3 H!thymidine incorporation is calculated by the following equation, where the values for no inhibition and maximal inhibition come from the medium controls, and the highest concentration of calicheamicin N-acetyl gamma, respectively: ##EQU3##

The percent inhibition values are plotted against sample concentrations, and from the resulting curves the sample concentration that gives 50% inhibition of 3 H!thymidine incorporation (IC50) is interpolated. For P67.6, A33, and m-CT-M-01 conjugates, the specificity of a particular conjugate for antigen-positive cells is calculated by taking the ratio of the IC50 against non-target cells to the IC50 against target cells. The same ratio is calculated for the free drug. Then, to correct for inherent differences in the sensitivities of the two cell lines to the drug, the Specificity Index for each sample is calculated as follows: ##EQU4##

For conjugates of Anti-Tac or h-CT-M-01, the Specificity Index is calculated as the ratio of IC50 'S for the non-targeting conjugate and the targeting conjugate as follows:

Human tumors (either ˜107 -108 cells or 5 to 8 fragments of solid tumors 2 mm3 in size) are implanted subcutaneously into athymic mice (nude mice) and test samples are inoculated intraperitoneally (ip) at several dose levels on a q 4 day×3 schedule, starting 2-3 days after tumor implantation with 5 mice per test group and 10 in the saline control group. Tumor mass is estimated by measuring the tumor length and width once weekly up to 42 days post tumor implantation with a Fowler ultra CAL II electronic caliper and using the formula: mg tumor={Length(mm)×Width(mm)}/2. Tumor growth inhibition is calculated as the ratio of the mean tumor mass of treated animals compared with untreated controls and is expressed as "% T/C". (0% T/C implies no detectable tumor. All control animals routinely develop easily measurable tumor.)

EX VIVO INHIBITION OF COLONY FORMATION

For P67.6 conjugates, human leukemic bone marrow cells which are CD-33 positive are plated in the presence of 2 ng/mL drug equivalents. The number of colonies which form are counted and reported as the percent versus a control which consists of a h-CT-M-O1 conjugate which does not recognize the CD-33 antigen. All the data reported were generated with bone marrow from one patient whose leukemic cells had good antigen expression and good response to this general type of treatment.

For anti-Tac, peripheral blood from CML patients was tested. Progenitor cells for cells of the various hematopoietic lineages can be detected by culturing bone marrow cells and blood cells in a semisolid matrix such as methylcellulose and observing the formation of colonies containing mature differentiated cells. There are progenitor cells that proliferate to form colonies of granulocytes or macrophages, or both, called colony-forming units for granulocytes-macrophages (CFU-GM). Some CFU-GM form colonies within seven days (D7 CFU-GM); some require fourteen days for colony formation (D14 CFU-GM) N. Jacobsen, et al., "Blood" 52: 221, (1978), and Ferrero D et al."Proc. Natl. Acad. Sci. USA" 80: 4114, (1983)!. Inhibition of the growth of D14 CFU-GM on blood cells treated with anti-Tac was compared to those treated with non-targeting MOPC 21 conjugates. The number of D14 CFU-GM colonies are plotted against sample concentrations, and from the resulting curves the sample concentration that gives 50% inhibition of D14 CFU-GM colony growth is interpolated. Specificity was measured by the ratio of the IC50 of the non-targeting conjugate versus the IC50 of the targeting conjugate. Normal blood does not produce CFU-GM colonies and normal bone marrow D14 CFU-GM colonies are not inhibited by anti-Tac conjugates.

The invention is further described with the following non-limiting preparations and examples. (Preparations describe the syntheses of compounds useful in this invention but for which there is known prior art. Examples describe the syntheses of compounds which are useful and new to this invention.)

A solution of 300 mg (1.68 mmol) of 3-(2-propenyloxy)benzoic acid in 5 mL of methylene chloride is cooled to -78° C. Ozone is introduced by bubbling the gas into the solution through a glass tube until a blue color persists. The solution is then purged with a stream of argon and 1 mL of methyl sulfide is added. The solution is diluted with 20 mL of ether and washed with water. The organic layer is separated and allowed to stand over magnesium sulfate then concentrated in vacuo to give 283 mg (93%) of 3-(2-oxoethoxy)benzoic acid as a colorless oil. The product is utilized in the next reaction without further purification: m.p. 120°-130°; the 1 H NMR (300 MHz, CDCl3) is consistent with the desired product; IR (KBr) 3400, 3000, 1680, 1590 cm-1 ; MS (CI low res) m/e 181 (M+H), 163, 139, 119.

A solution of 385 mg (1.63 mmol) of 4-(3-formylphenoxy)butanoic acid, ethyl ester and 850 mg (6.15 mmol) of potassium carbonate is stirred in 6 mL of methanol/water (3:2) at room temperature for 8 hours. The solution is then concentrated in vacuo. The residue is dissolved in 10 mL of 0.1N sodium hydroxide solution and washed with 20 mL of ether. The aqueous layer is separated and acidified with sodium bisulfate and extracted with ethyl acetate. The organic layer is washed with saturated sodium chloride solution, then dried over magnesium sulfate. The mixture is then filtered and concentrated in vacuo to give 315 mg of 4-(3-formylphenoxy)butanoic acid as a white solid. The product is utilized in the next reaction without further purification. m.p. 62°-63°; the 1 H NMR (300 MHz, CDCl3) is consistent with the desired product; IR (KBr) 3400, 3000, 1700, 1690, 1590 cm-1 ; MS (CI low res) m/e 209 (M+H), 191, 123.

A mixture of 253 mg (1.44 mmol) of 4-formylcinnamic acid and 32.61 mg of platinum oxide in 10 mL of methanol is stirred overnight at room temperature under an atmosphere of hydrogen supplied by a balloon. The mixture is filtered through celite and concentrated in vacuo. The residue is dissolved in 0.1N sodium hydroxide solution and washed with ether. The aqueous layer is then acidified and the product is extracted with ethyl acetate. The organic layer is washed with saturated sodium chloride solution and dried over magnesium sulfate. The solvent is removed in vacuo to afford an inseparable mixture of 4-formylphenylpropanoic acid and other reduction products. The mixture is utilized in the next reaction without characterization or further purification.

4'-Piperazinoacetophenone (102 mg) is dissolved in 1 mL of N,N-dimethylformamide. After addition of methyl 5-bromovalerate (0.077 mL) and potassium carbonate (69 mg), the mixture is stirred at room temperature for 65 hours. TLC (10% MeOH/CH2 Cl2) should show a single product spot without residual starting material. The reaction solution is evaporated under vacuum. The residue is taken up in methylene chloride, washed twice with water and dried over sodium sulfate. Evaporation of the solvent yields 137 mg of 4-(4-acetylphenyl)-1-piperazinevaleric acid, methyl ester as yellow crystals whose 1 H-NMR (CDCl3) spectrum is consistent with the assigned structure.

Under dry condition, 3.58 g (18.41 mmol) of 5-acetylsalicylic acid, methyl ester is dissolved in 25 mL of dry N,N-dimethylformamide. To this solution is added 3.07 g (20.58 mmol) of 5-bromo-1-pentene, 6.83 (20.58 mmol) of potassium carbonate, and 0.246 g (1.65 mmol) of potassium iodide, and the reaction mixture is stirred for 24 hours at ambient temperature. Another portion of 5-bromopentene is added to the reaction, followed by one-half portions of the other two reagents above, and stirring is continued for 72 hours. The mixture is then evaporated under high vacuum at 70° C. The residue is partitioned between ether/water and the organic phase is separated, dried with magnesium sulfate, filtered, and evaporated under vacuum to leave 4.60 g (95%) of 5-acetyl-2-(4-pentenyloxy)benzoic acid, methyl ester as a yellow liquid: IR (neat) 1735, 1710, 1680 cm-1 ; 1 HNMR (CDCl3) is consistent with the desired product; MS (FAB) mle 263 (M+ +H). Analysis calculated for C15 H18 O4 : C, 68.69; H, 6.92; O, 24.40. Found: C, 68.60; H, 6.92; O, 24.46.

A sample of 0.203 g (0.775 mmol) of 5-acetyl-2-(4-pentenyloxy)benzoic acid, methyl ester is dissolved in 5 mL of methylene dichloride, under an argon atmosphere, and cooled to -78° C. in a dry ice acetone bath, with stirring. Next, ozone gas is passed through this solution for 10 min, until it turns a light bluish color. Then 0.5 mL of dimethyl sulfide is added to quench the reaction and it is allowed to warm to room temperature for 2 hours. The mixture is then evaporated under high vacuum, leaving the crude aldehyde product as an oil which is used "as is" for the second step. It is dissolved in 5 mL of N,N-dimethylformamide, and 1.02 g (2.71 mmol) of pyridinium dichromate is added. This reaction mixture is sealed and allowed to stand for 20 hours. It is next poured into 50 mL of water, extracted with ether, and the organic phase is washed with water again, dried with magnesium sulfate, filtered, and evaporated, which gives oily crystals. These are recrystallized from a mixture of ethyl acetate and hexane, producing 0.109 g (50%) of 5-acetyl-2-(3-carboxypropoxy)benzoic acid, methyl ester as white crystals: m.p. 111°-113° C.; IR (KBr) 1725, 1645 cm-1 ; 1 HNMR (CDCl3) is consistent with the desired product; MS (FAB) m/e 281 (M+ +H). Analysis calculated for C14 Hl6 O6 : C, 60.00; H, 5.75; O, 34.25. Found: C, 59.96; H, 5.75; O, 34.27.

A solution of 2-fluoro-4-methoxyacetophenone in 5 mL of DMSO is stirred at 100° C. in the presence of 730 mg (15 mmol) of sodium cyanide to give a dark viscous sludge. The mixture is allowed to cool, then poured into 50 mL of ice water and acidified with 6N aqueous HCl. The acidic solution is extracted with ethyl acetate (50 mL×2) and the organic layers are combined and washed with water. The organic layer is then extracted twice with 1.0N aqueous sodium hydroxide solution. The basic layer is washed once with ether, then acidified with solid sodium bisulfate and extracted with ethyl acetate twice. The ethyl acetate layers are combined, then washed with 10% sodium bisulfate solution and saturated sodium chloride solution. The organic phase is dried over magnesium sulfate and concentrated in vacuo at ambient temperature to give 143 mg (31%) of an oil.

A solution of 41 mg (0.12 mmol) of 1- 10-(6-hydroxyhexyl)-10H-phenothiazin-2-yl!ethanone in 0.16 mL of N,N-dimethylformamide is treated with 158 mg (0.42 mmol) of pyridinium dichromate and stirred at room temperature for 12 hours. The mixture is diluted with ether and filtered through a pad of celite with the aid of 100 mL of ether. The filtrate is washed successively with 10% sodium bisulfate solution and saturated sodium chloride solution, then dried over magnesium sulfate and concentrated in vacuo to give 10 mg (23%) of 2-acetyl-10H-phenothiazine-10-hexanoic acid as a dark residue. The 1 H NMR (300 MHz, CDCl3) is consistent with the desired product. MS (CI) m/e 323 (M+ +H).

A sample of 0.140 g (0.50 mmol) of 5-acetyl-2-(3-carboxypropoxy)-benzoic acid, methyl ester (Example 19) is heated on a steam bath, under dry conditions with 5.49 mL (50.0 mmol) of N,N-dimethylethylenediamine for 5 hours. The mixture is allowed to cool for 20 hours to ambient temperature and evaporated under vacuum at 55° C. The brown gum produced is triturated with ether, and the remaining residue taken up in water and acidified with hydrochloric acid. This is then extracted with ethyl acetate, and the aqueous solution is evaporated under vacuum, leaving a gum. It is next triturated with hot chloroform and this solution is evaporated to give a brown glass. This is chromatographed on a preparatory silica gel plate which is eluted with a 9/1 mixture of chloroform to methanol. The product band is cut from the plate, triturated with the above solvent mixture, filtered, and evaporated leaving 0.025 g (15%) of 5-acetyl-2-(3-carboxypropoxy)-N-(2-dimethylaminoethyl)-benzamide as a light brown gum: MS (FAB) m/e 337.1753 Δ=+0.9 mmμ (M+ +H), 359 (M+ +Na). 1 HNMR (CDCl3) is consistent with the desired product.

The above product is dissolved in 2 mL of tetrahydrofuran and treated with an excess of 1N sodium hydroxide for 16 hours at ambient temperature. The organic cosolvent is removed under vacuum and the aqueous solution which remains is acidified with 1N HCl to a pH of about 5. The solution is then evaporated under vacuum to give a glass which crystallizes on standing. The resultant 5-acetyl-2-(3-carboxypropoxy)-N-(2-trimethylaminoethyl)benzamide, internal salt can be used without further purification. MS (FAB) m/e 351 (M+ +H).

The above compound is dissolved in ˜5 mL of methanol. Five equivalents of sodium hydroxide is added as a 5N solution in water. After 5 hours at ambient temperature the pH is adjusted to ˜7.5 with dilute HCl and the volatile components are removed under vacuum to give a crude product containing 5-acetyl-2- N-(2-trimethylaminoethyl)-3-carboxamidopropoxy!benzoic acid, internal salt. MS (CI) m/e 323 (M+ +H).

SYNTHESIS OF STRUCTURES B (Scheme 1)

General Procedure

The drug-hydrazide derivative (Q-Sp-S-S-W wherein Q=H2 NHN--) is dissolved in alcohol or other compatible organic solvent containing ˜3 to 10 equivalents of the carbonyl linker and ˜1-10% acetic acid or other appropriate acid catalyst. A minimal amount of solvent gives a faster reaction. Anhydrous conditions give the best results as the condensation is an equilibrium reaction. The reaction is allowed to proceed at a temperature of ˜20°-60° C. until complete by HPLC or alternately by TLC. This requires from a few hours to a day or more depending on the linker and the specific reaction conditions. The solvents are removed in vacuo and the crude product is purified on an appropriate silica gel, such as BIOSIL-A™, using an appropriate solvent system, such as a gradient of 0 to 20% methanol in either chloroform or ethyl acetate. The products are sufficiently pure for subsequent steps.

The carboxylic acid-hydrazones as obtained above are converted to the OSu esters (Z3 =N-succinimidyloxy) by dissolving them in an appropriate solvent such as acetonitrile or acetonitrile containing 10-20% N,N-dimethylforamide or tetrahydrofuran for better solubilization and adding ˜2-5 equivalents of N-hydroxysuccinimide and ˜2-10 equivalents of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) as the hydrochloride salt. The reaction is allowed to proceed at ambient temperature until complete as measured by HPLC or alternately by TLC, which is usually 1 to 8 hours. The solvents are then removed and the crude product is purified on an appropriate silica gel, such as BIOSIL-A™, using an appropriate solvent system, such as a gradient of 0 to 20% methanol in either chloroform or ethyl acetate. The products are then sufficiently pure for the conjugation step.

The activated ester from above is dissolved in an appropriate organic solvent, such as dimethylformamide, and added to a solution of antibody at ˜1-15 mg/mL in an appropriate buffer, such as pH 7.4 phosphate (50 mM, 100 mM salt) such that the concentration of organic co-solvent is ˜10-30% and ˜2-10 equivalents of active ester are used per mole of antibody. The conjugation reaction is allowed to proceed at ambient temperature for ˜4-24 hours. The solution is concentrated by use of a semipermeable membrane, if necessary, and purified by standard size-exclusion chromatography, such as with SEPHACRYL S™-200 gel. The monomer fractions are pooled and the loading of drug on the antibody is estimated by UV-VIS absorbance at 280 nm for antibody and 333 nm or other appropriate wavelength for the calicheamicin hydrazones.

SYNTHESIS OF STRUCTURES E (Scheme 2)

General Procedure

The carboxylic acids of the spacers are activated as the OSu esters (Z3 =N-succinimidyloxy) by dissolving them in an appropriate solvent such as tetrahydrofuran containing 10-20% dimethylformamide and adding ˜2-3 equivalents of N-hydroxysuccinimide and ˜2-5 equivalents of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) as the hydrochloride salt. The reaction is allowed to proceed at ambient temperature until complete as assessed by TLC, which is usually 1 to 8 hours. The solvents are then removed and the crude product is purified on an appropriate silica gel, such as BIOSIL-A™, using an appropriate solvent system, such as a gradient of 0 to 5% methanol in chloroform. The products are generally purified further by recrystallization from a mixture of ethyl acetate-hexanes or other appropriate solvents.

The activated ester from above is dissolved in an appropriate organic solvent, such as N,N-dimethylformamide, and added to a solution of antibody at ˜1-15 mg/mL in an appropriate buffer, such as pH 7.4 phosphate (50 mM, 100 mM salt) such that the concentration of organic co-solvent is ˜10-25% and ˜2-20 equivalents of active ester are used per mole of antibody. The conjugation reaction is allowed to proceed at ambient temperature for ˜4-24 hours. The buffer is exchanged and the organic co-solvents and by-products are removed by use of a desalting column such as a PD-10 using pH 5.5 acetate buffer (25 mM acetate, 100 mM NaCd). The solution is concentrated by use of a semipermeable membrane, if necessary, and the product is used without further purification for the following step. The number of carbonyl groups incorporated per antibody is usually about half the number of equivalents of OSu ester used and can be further quantified by use of p-nitrophenyl hydrazine or other comparable method, if desired.

SYNTHESIS OF STRUCTURES D (Method B-Scheme 2)

General Procedure

The drug hydrazide derivative is dissolved in an appropriate organic solvent, such as N,N-dimethylformamide, and added to a solution of antibody-linker conjugate (structure F) from the previous step at ˜1-15 mg/mL in an appropriate buffer, such as pH acetate (25 mM, 100 mM salt) such that the concentration of organic co-solvent is ˜10-15% and ˜2-15 equivalents of hydrazide are used per mole of antibody. The conjugation reaction is allowed to proceed at ambient temperature for ˜4-24 hours. The buffer is exchanged and the organic co-solvents and by-products are removed by use of a desalting column such as a PD-10 using pH 7.4 buffer (50 mM phosphate, 100 mM NaCl). The solution is concentrated by use of a semipermeable membrane, if necessary, and purified by standard size-exclusion chromatography, such as with SEPHACRYL™ S-200 gel. The monomer fractions are pooled and the loading of drug on the antibody is estimated by UV-VIS absorbence at 280 nm for antibody and 333 nm or other appropriate wavelength for the calicheamicin hydrazones.

The described conjugates are useful for inhibiting the growth of unwanted cells which is an important part of the invention. Accordingly, the invention also includes pharmaceutical compositions, most preferably a parenteral composition suitable for injection into the body of a warm-blooded mammal. Such compositions are formulated by methods which are commonly used in pharmaceutical chemistry. The conjugates are acceptably soluble in physiologically-acceptable fluids, such as physiological saline solutions and other aqueous solutions which can safely be administered parenterally.

Products for parenteral administration are often formulated and distributed in solid, preferably freeze-dried form, for reconstitution immediately before use. Such formulations are useful compositions of the present invention. Their preparation is well understood by pharmaceutical chemists; in general, they comprise mixtures of inorganic salts, to confer isotonicity, and dispersing agents, such as sucrose, to allow the dried preparation to dissolve quickly upon reconstitution. Such formulations are reconstituted with highly purified water or physiologically acceptable buffers to a known concentration, based on the drug. A preferred freeze-dried pharmaceutical composition for inhibiting the growth of cells is obtained by freeze-drying an approximately 1 mg/ml solution of the conjugate dissolved in about 5 mM sodium phosphate buffer at a pH of about 7.4 containing about 100 mM sodium chloride and about 100 mM sucrose. For the conjugate, which has the formula Z3 CO-Alk1 -Sp1 -Ar-Sp2 -Alk2 -C(Z1)=Z2 !m, Z3 is preferably antibody h-CT-M-01 or h-p67.6; Alk1 is preferably C4 alkylene; Sp1 is preferably --O--; Ar is preferably 1,4-phenylene; Alk2 and Sp2 preferably are together a bond; Z1 is preferably C1 alkyl; and Z2 is preferably calicheamicin N-acetyl gamma dimethyl hydrazide.

The optimum dosage and administration schedule of conjugates of the invention must be determined by the treating physician, in light of the patient's condition.

It is customary, of course, to administer cytotoxic drugs in the form of divided doses, with intervals of days or weeks between each series of doses. The conjugates are effective over a wide dosage range, and dosages per week will usually fall within the range from about 1 to about 10,000 μg/m2 of drug, more preferably in the range from about 10 to about 200 μg/m2.